The HER-2/neu (erbB-2) oncogene encodes a receptor tyrosine kinase (RTK) that has been extensively investigated because of its role in several human carcinomas (Hynes and Stern, 1994, Biochim. et Biophys. Acta 1198:165-184; and Dougall et al., 1994, Oncogene 9:2109-2123) and in mammalian development (Lee et al., 1995, Nature 378:394-398). Her-2 (also referred to as “erbB2,” “p185” or “c-neu”) is a 185 kDa member of the epidermal growth factor (EGF) receptor tyrosine kinase family. The sequence of the Her-2 protein was determined from a cDNA that was cloned by homology to the epidermal growth factor receptor (EGFR) mRNA from placenta (Coussens et al. 1985, Science 230:1132-1139) and from a gastric carcinoma cell line (Yamamoto et al., 1986, Nature 319:230-234). The full-length HER-2 mRNA encodes a transmembrane glycoprotein of 185 kDa in normal and malignant human tissues (p185HER-2) (Hynes and Steen, 1994, Biochim. et Biophys. Acta 1198:165-184; and Dougall et al., 1994, Oncogene 9:2109-2123).
The function of the HER-2 gene has been examined mainly by expressing the cDNA corresponding to the 4.5 kb transcript in transfected cells and from the structure and biochemical properties of the 185 kDa protein product. P185HER-2 consists of a large extracellular domain, a transmembrane segment, and an intracellular domain with tyrosine kinase activity (Hynes and Stern, 1994, Biochim. et Biophys. Acta 1198:165-184; and Dougall et al., 1994, Oncogene 9:2109-2123). Overexpression of p185HER-2 causes phenotypic transformation of cultured cells (DiFiore et al., 1987, Science 237:178-182; and Hudziak et al., 1987, Proc. Natl. Acad. Sci. USA 84:7159-7163) and has been associated with aggressive clinical progression of breast and ovarian cancer (Slamon et al., 1987, Science 235:177-182; and Slamon et al., 1989, Science 244:707-712).
p185HER-2 is highly homologous to the EGFR. However, a ligand that directly binds with high affinity to p185HER-2 has not yet been identified. Moreover, the signaling activity of HER-2 may be mediated through heterodimerization with other ligand-binding members of the EGFR family (Carraway and Cantley, Cell 78:5-8, 1994; Earp et al., 1995, Breast Cancer Res. Treat. 35:115-132; and Qian et al., 1995, Oncogene 10:211-219).
Divergent proteins, containing regions of the extracellular domains of HER family RTKs, are generated through proteolytic processing of full length receptors (Lin and Clinton, 1991, Oncogene 6:639-643; Zabrecky et al., 1991, J. Biol. Chem. 266:1716-1720; Pupa et al., 1993, Oncogene 8:2917-2923; Vecchi et al., 1996, J. Biol. Chem. 271:18989-18995; and Vecchi and Carpenter, 1997, J. Cell Biol, 139:995-1003) and through alternative RNA processing (Petch et al., 1990, Mol. Cell. Biol. 10:2973-2982; Scott et al., 1993, Mol. Cell. Biol. 13:2247-2257; and Lee and Maihle, 1998, Oncogene 16:3243-3252).
The extracellular domain of p185HER-2 (hereinafter referred to as “ECD”) is proteolytically shed from breast carcinoma cells in culture (Petch et al., 1990, Mol. Cell. Biol. 10:2973-2982; Scott et al., 1993, Mol. Cell. Biol. 13:2247-2257; and Lee and Maihle, 1998, Oncogene 16:3243-3252), and is found in the serum of some cancer patients (Leitzel et al., 1992, J. Clin. Oncol. 10:1436-1443) where it is may be a serum marker of metastatic breast cancer (Leitzel et al., 1992, J. Clin. Oncol. 10:1436-1443) and may allow escape of HER-2-rich tumors from immunological control (Baselga et al., 1997, J. Clin. Oncol. 14:737-744, Brodowicz et al., 1997, Int. J. Cancer 73:875-879). While shed Her-2 ECD serum levels correlate with tumor mass, additional studies have demonstrated that shed Her-2 ECD serum levels represent an independent marker of poor clinical outcome in patients with Her-2 overexpressing metastatic breast cancer (Ali et al., 2002, Clin. Chem. 48:1314-1320; Molina et al., 2002, Clin. Cancer Res. 8:347-353).
A truncated extracellular domain of HER-2 is also the product of a 2.3 kb alternative transcript generated by use of a polyadenylation signal within an intron (Scott et al., 1993, Mol. Cell. Biol. 13:2247-2257). The alternative transcript was first identified in the gastric carcinoma cell line, MKN7 (Yamamoto et al., 1986, Nature 319:230-234; and Scott et al., 1993, Mol. Cell. Biol. 13:2247-2257) and the truncated receptor was located within the perinuclear cytoplasm rather than secreted from these tumor cells (Scott et al., 1993, Mol. Cell. Biol. 13:2247-2257). However, no particular therapeutic, diagnostic or research utility has been ascribed to this truncated extracellular domain polypeptide. A truncated extracellular domain of the EGFR, generated by alternative splicing (Petch et al., 1990, Mol. Cell. Biol. 10:2973-2982) is secreted, exhibits ligand-binding as well as dimerization properties (Basu et al., 1989, Mol. Cell. Biol. 9:671-677), and may have a dominant negative effect on receptor function (Basu et al., 1989, Mol. Cell. Biol. 9:671-677; and Flickinger et al., 1992, Mol. Cell. Biol. 12:883-893).
An additional alternatively spliced product of Her-2, designated herstatin, has also been identified (Doherty et al., 1999, Proc. Natl. Acad. Sci. 96:10869-10874; Azios et al., 2001, Oncogene 20:5199-5209; Justman and Clinton, 2002, J. Biol. Chem. 277:20618-20624). This protein consists of subdomains I and II from the extracellular domain followed by a unique C-terminal sequence encoded by intron 8. Herstatin is secreted and binds with nanomolar affinity to EGFR family members. It appears to function as an autoinhibitor by disrupting receptor dimerization and receptor phosphorylation, resulting in inhibition of AKT signaling and suppression of proliferation in EGFR or Her-2 overexpressing cells. Herstatin was shown to be present at reduced levels in Her-2 overexpressing tumor cells, suggesting that if Herstatin plays a role in regulating normal cell growth, this is circumvented in tumor cells.
Signaling by the EGF family of receptors is initiated by ligand binding which triggers homo- or hetero-receptor dimerization, reciprocal tyrosine phosphorylation of the cytoplasmic tails, and activation of intracellular signal transduction pathways. The biologic consequence of EGF receptor signaling is frequently associated with cellular differentiation, growth or survival.
Overexpression of Her-2 occurs in 25-30% of breast cancers. Patients with Her-2 overexpressing tumors have a distinctly unfavorable clinical course, characterized by shortened time to disease recurrence and reduced survival. The precise mechanisms responsible for this association are not established. In this regard, it has been suggested that the overexpressed Her-2 receptor can more readily heterodimerize with other EGF receptor family members that have bound ligands, thereby initiating intracellular signaling cascades leading to growth and resistance to apoptosis. Alternatively, the high copy number of Her-2 receptors on the tumor cell surface can promote ligand independent receptor homodimerization, and intracellular signaling leading to tumor cell growth and survival.
Another mechanism that may account for poor clinical outcome in Her-2 overexpressing tumors is suggested by the observation that, in some Her-2 overexpressing tumor cells, the receptor is processed by an unknown metalloprotease (or metalloproteinase) to yield a truncated, membrane-associated receptor (sometimes referred to as a “stub” and also known as p95), and a soluble extracellular domain (also known as ECD, ECD105, or p105).
As with other EGF receptor family members, loss of the extracellular ligand binding domain renders the Her-2 intracellular membrane-associated domain a constitutively active tyrosine kinase. It has therefore been postulated that the processing of the Her-2 extracellular domain creates a constitutively active receptor that can directly deliver growth and survival signals to the cancer cell. In this regard, it has been shown that an engineered version of Her-2, lacking the extracellular domain, is 10-100 fold more efficient than the full length receptor in cellular transformation assays.
Moreover, the truncated form of Her-2 receptor (p95) has been shown to interact with and potently activate signaling through the EGF receptor (also referred to as “Her-1”). Most compelling, immunohistochemical analysis of clinical breast cancer specimens strongly suggest that poor clinical outcome is more closely associated with the presence of the truncated Her-2 receptor (p95) rather than the intact receptor (p185) in the tumor cell as discussed by Clinton (U.S. Pat. No. 6,541,214).
The ADAM (A Disintegrin And Metalloprotease) family of proteases has been demonstrated to catalyze cell surface ecto domain shedding of specific proteins (Moss and Lambert, 2002, Essays in Biochemistry 38:141-153; Chang and Werb, 2001, Trends in Cell Biology 11:537-543, Seals and Courtneidge, 2003, Genes and Development 17:7-30). The domain structure of ADAM family members places the protease catalytic domain of these type I membrane proteins extracellularly. From the amino terminus of the protein, the domains include a pro domain, catalytic domain, disintegrin domain, cysteine rich region and EGF repeat followed by the transmembrane domain and cytoplasmic tail. The pro domain is processed to form the mature proteolytically active form. The disintegrin domain may be involved in adhesion or substrate recognition and binding.
ADAM 10 was the first ADAM family member shown to have proteolytic activity. It has been demonstrated to cleave proteins such as APP and Notch as well as other cell surface proteins. Of interest, substrates for ADAM 10 include HB-EGF, a member of the epidermal growth factor family, which is important in cell transformation and mitogenesis. Cleavage of the extracellular domain of HB-EGF leads to the generation of a soluble fragment of HB-EGF that binds to and activates the EGF receptor. ADAM 15 has also been demonstrated to be an active protease and has been shown to degrade both type IV collagen and gelatin. In addition, ADAM 15 has been shown to actively participate in binding to integrins, including alpha5beta and alphavbeta3, through its disintegrin domain.
In Her-2 overexpressing cells, Her-2 had been found to undergo cleavage to form p95 in the presence of 4-aminophenylmercuric acetate (APMA), a well-known metalloprotease activator (Molina et al., 2001, Cancer Res. 61:4744-4749). APMA-mediated cleavage of Her-2 to form p95 was found to be inhibited in the same cells in the presence of batimastat, a broad-spectrum metalloprotease inhibitor. Additionally, Herceptin™ (also referred to as trastuzumab), an anti-Her-2 monoclonal antibody that has shown clinical efficacy in Her-2 overexpressing breast cancer, has been shown to inhibit enzymatic cleavage of intact Her-2 (p185) into an ECD portion and the p95 constitutively active kinase membrane-associated portion in vitro. It has been suggested that this cleavage-inhibitory effect of Herceptin™ may be mediated by antibody binding near the enzymatic clip site thereby interfering with cleavage enzyme-substrate interaction via steric hindrance. However, the mechanism by which Herceptin acts in the body (e.g., in vivo) remains unclear. Studies have shown that Herceptin appears to have multiple cellular functions that serve to inhibit Her-2 oncogenic signaling through different mechanisms (see, e.g., Baselga, et al., Seminars in Oncology, 2001, 28(5), suppl. 16, pp 4-11 and Baselga et al., Annals of Oncology, 2001, 12, suppl. 1, pp S35-S41). While some studies propose that at least one of the mechanisms of action of Herceptin is related to inhibition of Her-2 shedding, other studies, in fact, show that shedding continues to occur in patients treated with the antibody (Pegram et al., Journal of Clinical Oncology, 1998, 16(8), 2659-2671).
Despite the prevalence, morbidity and mortality associated with breast cancer, and other Her-2 overexpressing malignancies, there are few, if any, effective therapies for these diseases and disorders. Thus, there is an acute need for treatments and therapeutics for Her-2 overexpressing breast, and other, cancers, and the present invention meets this and other needs.